Bottom Line:
Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells.Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells.The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1).

ABSTRACTWe characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.

Figure 2: Antagonist sensitivity of the AChR currents activated in IMR-32 cells. (A) The concentration response relationship is shown for d-tubocurare (d-TC; ▪), mecamylamine (Mec; ▾), and hexamethonium (C-6; •) on IMR-32 cells with IC50 values of 0.4 ± 0.2, 3.2 ± 0.6, and 8.5 ± 3.0 μM, respectively. All drugs were coapplied with 100 μM ACh at a holding potential of −60 mV. The peak amplitudes of the resulting currents were then plotted against concentration of the antagonist after normalizing to the peak amplitude of the current recorded in the absence of any antagonist. All three drugs completely blocked the currents at higher concentrations. (B) Representative currents are shown for testing the sensitivity of IMR-32 AChR currents to the reversible α7 selective antagonist MLA (20 nM). No inhibition was observed, which was consistent with the idea that the macroscopic current recorded in the cells reflects predominately the activity of α3 AChRs.

Mentions:
Responses to 100 μM ACh were antagonized by coapplication of the antagonists d-tubocurare, mecamylamine, and hexamethonium with IC50 values of 0.4 ± 0.2, 3.2 ± 0.6, and 8.5 ± 3.0 μM, respectively (Fig. 2 A). The reversible α7 selective antagonist, methyllycaconitine (MLA), had no effect on Nic-activated (300 μM) currents when applied at 20 nM with a 10 min preincubation (Fig. 2 B). This concentration of MLA (20 nM) was almost three orders of magnitude higher than the IC50 reported for homomeric α7 AChRs (Palma et al. 1996) or α7 containing AChRs in rat hippocampal neurons (Alkondon et al. 1992). The lack of antagonism by MLA was consistent with the fact that the currents lacked the rapid desensitization that is a hallmark property of α7 AChRs. To validate the ability of our application system to activate α7 AChRs efficiently before they desensitized, we recorded the rapidly desensitizing Nic-activated currents that have been attributed to α7 AChRs from rat hippocampal neurons (unpublished data). Thus, it was concluded that any contribution of α7 AChRs to macroscopic currents from IMR-32 cells was negligible.

Figure 2: Antagonist sensitivity of the AChR currents activated in IMR-32 cells. (A) The concentration response relationship is shown for d-tubocurare (d-TC; ▪), mecamylamine (Mec; ▾), and hexamethonium (C-6; •) on IMR-32 cells with IC50 values of 0.4 ± 0.2, 3.2 ± 0.6, and 8.5 ± 3.0 μM, respectively. All drugs were coapplied with 100 μM ACh at a holding potential of −60 mV. The peak amplitudes of the resulting currents were then plotted against concentration of the antagonist after normalizing to the peak amplitude of the current recorded in the absence of any antagonist. All three drugs completely blocked the currents at higher concentrations. (B) Representative currents are shown for testing the sensitivity of IMR-32 AChR currents to the reversible α7 selective antagonist MLA (20 nM). No inhibition was observed, which was consistent with the idea that the macroscopic current recorded in the cells reflects predominately the activity of α3 AChRs.

Mentions:
Responses to 100 μM ACh were antagonized by coapplication of the antagonists d-tubocurare, mecamylamine, and hexamethonium with IC50 values of 0.4 ± 0.2, 3.2 ± 0.6, and 8.5 ± 3.0 μM, respectively (Fig. 2 A). The reversible α7 selective antagonist, methyllycaconitine (MLA), had no effect on Nic-activated (300 μM) currents when applied at 20 nM with a 10 min preincubation (Fig. 2 B). This concentration of MLA (20 nM) was almost three orders of magnitude higher than the IC50 reported for homomeric α7 AChRs (Palma et al. 1996) or α7 containing AChRs in rat hippocampal neurons (Alkondon et al. 1992). The lack of antagonism by MLA was consistent with the fact that the currents lacked the rapid desensitization that is a hallmark property of α7 AChRs. To validate the ability of our application system to activate α7 AChRs efficiently before they desensitized, we recorded the rapidly desensitizing Nic-activated currents that have been attributed to α7 AChRs from rat hippocampal neurons (unpublished data). Thus, it was concluded that any contribution of α7 AChRs to macroscopic currents from IMR-32 cells was negligible.

Bottom Line:
Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells.Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells.The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1).

ABSTRACTWe characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.